G-protein coupled receptors represent a major focus of pharmaceutical drug discovery efforts, owing to their involvement in many important signaling pathways associated with both normal physiology and human disease. Nevertheless, little structural and functional information is known about these membrane proteins due to their recalcitrance to expression, and formation of soluble, monodisperse, functional membrane receptor preparations. However, the recent development of "nanodisc" technology should permit the functional reconstitution of soluble chemokine GPCRs for biophysical characterization. The reconstitution of other GPCRs into nanodiscs has been successful and allowed subsequent biophysical studies which have yielded important information about their activation mechanisms. These studies will establish a protocol for the preparation of soluble, monodisperse, and functional chemokine receptors for use in a wide array of biophysical experiments aimed at increasing our understanding of disease-related chemokine receptors. Specific Aims are as follows: (1) To further optimize the yield of overexpressed chemokine receptors. (2) To reconstitute and characterize the nanodisc technology. (3) To find conditions for the functional reconstitution of the chemokine receptor CCR1 into nanodiscs. (4) To elucidate the activation/inhibition mechanisms of CCR1 using fluorescence spectroscopy. CCR1 can currently be expressed at ~1 mg/L, a quantity that is sufficient for the proposed studies;however, higher expression levels are feasible through the use of a codon-optimized gene. After implementing the published nanodisc technology, conditions (detergents, lipids, etc.) will be screened for the functional reconstitution of chemokine receptors. We will measure the ability of reconstituted receptors to bind chemokines and activate G proteins to assess functionality. Subsequently, we will use fluorescence assays to monitor the agonist/antagonist induced conformational changes that occur in the chemokine receptor upon ligand binding. Describing these changes and correlating them with the physiochemical characteristics of the ligands used should highlight important features of the receptor:ligand interactions. These studies will contribute to our understanding of the mechanism of activation of chemokine receptors, an important class of proteins known to be involved in the pathologies of cancer, multiple sclerosis, rheumatoid arthritis, renal fibrosis, and transplant rejection. Because inappropriate chemokine-mediated cell migration has been shown to hasten the growth and metastasis of cancer, our long term goal is to use our understanding of receptor responses to find methods for inhibiting the proliferation of cancerous cells.